ANTICANCER RESEARCH 26: 4647-4652 (2006)
Up-regulated Expression of the Uridine Phosphorylase Gene in Human Gastric Tumors is Correlated with a Favorable Prognosis
KIYOKO KAWAMURA1, NOBUHIRO TAKIGUCHI2, AKIHIKO WADA1, HISANORI TAKENOBU1, HIDEKI KIMURA3, HIROAKI SODA2, MATSUO NAGATA2, TAKEHIDE ASANO2 and MASATOSHI TAGAWA1
Division of 1Pathology, 2Gastroenterological Surgery and 3Thoracic Disease, Chiba Cancer Center, 666-2 Nitona, Chuo-ku, Chiba 260-8717, Japan
Abstract. Uridine phosphorylase (UP) is one of the enzymes 4), into its nucleotides. It is however controversial as to involved in 5-fluorouracil (5-FU) activation. The expression which is the key enzyme for the activation of 5-FU and can was compared in paired specimens from cancerous and non- predict the antitumor effects (4-8). cancerous regions of gastric, colon and lung cancer patients. Expression of the UP gene in human solid tumors is Among 28 paired gastric samples, 20 cases showed greater elevated compared with that of the adjacent normal tissues expression in tumors than in normal surrounding tissues and (1, 9-11). Analyses of the regulatory region suggested that 8 cases showed equal or lower expression levels in tumors. All inflammatory cytokines and oncogenes could up-regulate the gastric patients received 5-FU before and/or after the the expression, while P53 decreased the expression (12, 13), surgical resection and the prognosis of the patients, whose UP although the contribution of such transcriptional regulation tumor expression increased, was relatively better than that of to the up-regulated expression in tumors remains unclear. the patients with equal or less UP gene tumor expression. In Moreover, the relationship between the increased contrast, most of the colon (22 cases in total) and all the lung expression in tumors and the sensitivity of the tumors to cancer patients (14 in total) did not receive 5-FU and the 5-FU has not been well-established in clinical settings (4, 8, majority of the colon (12 cases) and lung (10 cases) specimens 14). Although previous studies demonstrated that up- showed lower expression in the cancerous region. The regulated UP expression in breast and oral cancer was a differential expression between cancerous and non-cancerous poor prognostic marker (10, 11), the prognostic value has regions in colon and lung cancers was not linked with the not been reported in other types of cancer. In this study, the prognosis. These data suggest that the paired expression level UP gene expression was compared in paired gastric, colon of the UP gene in gastric cancer is a possible prognostic marker and lung specimens, derived from cancerous and non- for the patients who received 5-FU. cancerous regions, and the association between prognosis and the up-regulation was investigated. The expression was Uridine phosphorylase (UP, EC 2.4.2.3) is an enzyme examined with semi-quantitative reverse transcriptase- involved in the pyrimidine salvage pathway, catalyzing the polymerase chain reaction (RT-PCR), since UP expression is reversible phosphorolysis of pyrimidine nucleosides (1). UP not modified post-transcriptionally and accordingly, RT-PCR is present in most tissues and has an important role in the is a sensitive marker for the enzyme activity (4, 10). homeostatic regulation of plasma uridine concentrations that consequently influence a number of cellular functions Materials and Methods (1, 2). UP, as well as orotate phosphoribosyltransferase and thymidine phosphorylase, mediates the conversion of 5- Tissue specimen and RNA extraction. Surgically resected tumors and fluorouracil (5-FU), widely used as an anticancer agent (3, their adjacent normal tissues (more than 3 cm away from the tumors) were obtained from the Chiba Cancer Center Tissue Bank, including 28 gastric, 22 colon and 14 lung specimens. The use of the human tissues was approved by the Chiba Cancer Center Correspondence to: Dr. Masatoshi Tagawa, Division of Pathology, Ethical Committee. Grading and stage classifications were based Chiba Cancer Center Research Institute, 666-2 Nitona, Chuo-ku, on the classification and the rule of Japanese corresponding Chiba 260-8717 Japan. Tel: +81-43-264-5431 ext.5101, Fax: +81- societies, which are almost identical to the TNM classification of 43-265-4459, e-mail: [email protected] International Union Against Cancer. RNA was extracted with TRIzol reagent (Life Technologies, Rockville, MD, USA), Key Words: Uridine phosphorylase, gastric cancer, prognosis. according to the manufacturer’s protocol.
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RT-PCR analysis. First-strand cDNA was synthesized with total RNA using Superscript II reverse transciptase (Life Technologies) and random primers. Amplification of an equal amount of respective cDNA was performed for 27-35 cycles (UP) or 25 cycles (glyceraldehyde 3-phosphate dehydrogenase, GAPDH) with the following primers and conditions: for the UP gene, forward (5’- CCCAGCCTTGTTTGGAGATG-3’) and reverse (5’- CAGACCTATCCCACCAGAAG-3’) and 10 sec at 95ÆC for denaturation, 20 sec at 54ÆC for primer annealing, 30 sec at 72ÆC for primer extension; for the GADPH gene, forward (5'- ACCACAGTCCATGCCATCAC-3') and reverse (5'- TCCACCACCCTGTTGCTGTA-3'), and 15 sec at 94ÆC, 15 sec at 60ÆC, 40 sec at 72ÆC. The amplified PCR fragments were analyzed with agarose gel electrophoresis. The primer sets for the UP gene did not hybridize with a homologous UP gene (UP2) expressed in kidney, spleen and liver but not in human tumors and has broad substrate specificity (15).
Statistical analysis. Survival of the patients was conducted by the Kaplan-Meier test and statistical analysis was performed by the log- rank test. Figure 1. Expression of the UP and GAPDH genes as a control in paired Results specimens from cancerous and non-cancerous regions of the same patients. The number corresponds to the patient number in Table I. (A) lung, (B), gastric and (C) colon cancer. Expression of the UP gene in cancerous and non-cancerous specimens. The UP gene expression was examined in paired specimens from gastric, colon and lung cancers (Figure 1). The amounts of the first-strand cDNA were adjusted, based Prognosis and UP gene expression. The patients were divided on the GAPDH gene expression. The amplification cycles into two groups based on the UP gene expression: up- were different among the samples, but the same cycle regulated in tumors (T>N group in Table I) and non-up- number was used for respective paired samples. Expression regulated in tumors (T=N and T
4648 Kawamura et al: Expression of UP Gene in Gastric Tumors
Table I. Clinicopathological features of gastric, colon and lung cancers and the UP gene expression.
Case Age Gender Histologya Grade Stage Expressionb Case Age Gender Histologya Grade Stage Expressionb
Gastric 9* 74 F WDA T4N0M0 II T
samples of the same patients. The expression in gastric local cytokine milieu of tumors can thereby result in the up- cancer has not been reported and the mechanisms of the regulation. Since macrophages, infiltrated into tumors, were up-regulated expression in tumors are also currently positive for UP (11), the up-regulation could be influenced unknown. The up-regulation is presumed to be partly due by the degree of macrophage migration. to the increased DNA synthesis in tumors, which facilitates The results of the present study showed that the up- the nucleic acids metabolism, and to the activation of regulated UP gene was a favorable factor for gastric cancer oncogene and/or loss of P53 function, both of which can patients, but not for colon or lung cancer patients. Previous activate transcription (12, 13). Furthermore, the up- studies, however, indicated that up-regulation was regulated UP expression contributes to tumorigenesis by unfavorable for breast and oral cancer patients with high promoting anchorage-independent growth (16). incidence of metastasis (10, 11). Supposing that the up- Inflammatory cytokines, which can induce up-regulation regulation is linked with the proliferation activities of tumor (12), may contribute to up-regulation in tumor specimens. cells and infiltration levels of macrophages, the elevated Immune reactions are generated at a tumor site and the gene expression could be associated with a poor prognosis.
4649 ANTICANCER RESEARCH 26: 4647-4652 (2006)
prognosis linked with up-regulated UP gene expression may be due to the increased sensitivity of the patients to 5-FU. A previous study with colon cancer specimens disproved the relationship between UP gene expression and 5-FU susceptibility (4); however, tissue concentrations of 5-FU after administration and absolute expression levels of the UP gene expression should be different among organs. The discrepant results regarding UP expression and the drug sensitivity could be due to the tissue differences between colon and stomach. Administration of 5-FU itself did not induce up-regulation of the UP gene (14) and the present data in colon cancer patients supported this report; therefore, the up-regulated expression is irrelevant to the chemotherapy administered to the gastric cancer patients. In summary, we found that the up-regulated UP gene in tumors and its clinical value were dependent on cancer types. This is the first report, to our knowledge, to suggest that the up-regulation of the UP gene is a possible prognostic marker for gastric cancer patients with 5-FU treatment. Since other 5-FU derivatives, where the role of UP is minimal, are currently used as chemotherapeutic agents, our assumption will be testified based on the gastric cancer patients who received the 5-FU derivatives. An additional investigation of 5-FU-naïve patients is required as well, although such specimens are not currently available in our Tissue Bank.
Acknowledgements
We thank Prof. Shulin Li (School of Veterinary Medicine, Louisiana State University, USA) and Dr. Zhengmao Zhang for valuable information and technical assistance, respectively. This work was supported by a grant-in-aid for the COE (Center of Excellence) research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, a grant-in-aid for scientific research from the Japan Society for the Promotion of Science, a grant-in-aid from the Tsuchiya Foundation and the Futaba Electronics Memorial Foundation.
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